HRAS Research Articles

Analysis of β-catenin gene mutations and gene expression in liver tumours of C57BL/10J mice produced by chronic administration of sodium phenobarbital

In this study liver tumours produced in male and female mice of the low spontaneous liver tumour incidence C57BL/10J strain treated for 99 weeks with 1000 ppm in the diet with the model constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were analysed for β-catenin mutations by Western immunoblotting and DNA/RNA analysis. Some gene array analysis was also performed to identify genes involved in CAR activation and in β-catenin and Hras gene mutations. Analysis of 8 male and 2 female NaPB-induced liver tumour samples (comprising 2 adenomas, 6 carcinomas and 2 samples containing separate adenomas and carcinomas) revealed truncated β-catenin forms in just 4 male liver tumour samples, with the presence of the truncated β-catenin forms being confirmed by β-catenin exon 1–3 mutation analysis. Microarray gene expression analysis was performed with three of the NaPB-induced male mouse liver tumour samples where β-catenin mutations had not been identified by Western immunoblotting and DNA/RNA analysis and with three liver samples from both NaPB-induced non-tumour tissue and control animals. Treatment with NaPB resulted in induction of Cyp2b subfamily gene expression in both NaPB-induced mouse liver tumours and in NaPB-treated non-tumour tissue. In addition, the gene expression analysis demonstrated that the β-catenin and Hras pathways were not modified in NaPB-induced mouse liver tumours not exhibiting truncated β-catenin forms. Overall, while chronic administration of the model CAR activator NaPB results in both hepatocellular adenoma and carcinoma in the low spontaneous liver tumour incidence C57BL/10J mouse strain, only 40 % of the liver tumours evaluated in this study had β-catenin mutations. These results are in agreement with previous studies with the CAR activator oxazepam and demonstrate that mouse liver tumours induced by nongenotoxic CAR activators in the absence of initiation with a genotoxic agent are due to a number of mechanisms, including those largely independent of either the Wnt/β-catenin signalling pathway or Hras oncogene mutations.

A pathogenic variation of HRAS gene causing Costello syndrome: a Ras/MAPK pathway syndrome

1例Costello综合征患儿 女，3月龄，因体重下降就诊。经查体及相关基因检测发现患儿HRAS基因杂合变异：c.34G>A（p.G12S），此变异为已报道致病变异，但患儿父母及哥哥外周血样本中未见该变异，确诊为Costello综合征。.

Abstract PR09: Phase 1 clinical safety and efficacy of ASN007, a novel oral ERK1/2 inhibitor, in patients with RAS, RAF or MEK mutant advanced solid tumors

Abstract Background: The oncogenic mitogen-activated protein kinase (MAPK) pathway is dysregulated in a broad range of cancers through mutations in BRAF, KRAS, NRAS, HRAS and MEK1 genes. ASN007 is a novel, potent and orally bioavailable inhibitor of ERK1/2 kinases (IC50 1-2nM) with a very long target residence time (550 min). Preclinical studies show strong anti-tumor activity in multiple RAS mutant xenograft and PDX models, irrespective of mutation subtype. ASN007 also maintains strong activity in BRAF mutant melanoma PDX models resistant to BRAF and MEK inhibition. Preclinical activity is seen with both intermittent as well as daily dosing. This is the initial report of ASN007 clinical data from this first-in-human Phase 1 Study. Methods: This Phase 1 trial evaluates ASN007 in patients (PS 0-1) with metastatic solid tumors refractory to standard therapies using an accelerated dose titration design for once daily (QD) and once weekly (QW) oral dosing (NCT03415126). Primary objectives in Part A (dose finding) include safety, tolerability, PK and PD to determine the Maximum Tolerated Dose (MTD). Part B (disease specific expansion) will evaluate clinical activity in cohorts of 15 patients each with tumors harboring specific mutations which include NRAS, HRAS, MEK1 and BRAF. Results: To date, 42 eligible patients (pts) were enrolled at dose levels ranging from 10mg to 80mg QD and 80 mg to 350mg QW. The respective MTDs are 40mg QD and 250mg QW. Reversible treatment-related adverse events (TRAEs) at 40mg QD (n=10) included rash ( 90%, Gr3 10%), nausea/vomiting (Gr1 30%), diarrhea (Gr1 30%), fatigue (20%, Gr3 10%) and central serous retinopathy (CSR) (30%, no G3) and at 250mg QW (n=9) included rash (33%, no Gr3), CSR (11%, Gr3 11%), blurred vision (44%, Gr3 11%), nausea/vomiting (33%, Gr3 11%) and diarrhea (33%, no Gr3). Dose limiting toxicities (DLTs) included Gr3 CSR at 60 and 80 QD, Gr3 rash at 80mg QD and Gr3 AST at 350mg QW. Durable clinical benefit was noted for 3 pts treated with QW ASN007 at doses 120 mg to 250 mg: a confirmed partial response (-57%) in a patient with HRAS-mutant salivary gland cancer for 5+ months, stable disease in KRAS mutant ovarian cancer for 9+ months and stable disease in BRAF V600E mutant thyroid cancer for 8+ months. Pharmacokinetic studies demonstrated dose dependent increase in the Cmax and AUC24 with both QD and QW dosing. QW dosing Cmax levels exceed average IC50 values more than >30-fold. Inter-subject variability was moderate (≤ 50%) and the elimination t1/2 was 10-15 hours. Conclusions: ASN007 is a selective oral inhibitor of ERK1/2 with a long target residence time. QD or QW dosing is feasible with expected reversible and manageable AE profile and dose dependent exposure. The dose of 250mg QW was selected for further development due to favorable tolerability and encouraging signs of clinical activity. Part B of the study is open to enroll patients with solid tumors harboring BRAF, MEK1, and RAS mutations. Updated clinical, safety, PK/PD and efficacy data will be presented. Citation Format: Anthony W. Tolcher, Ryan J. Sullivan, Drew W. Rasco, Zeynep Eroglu, Nehal Lakhani, Dana Kessler, Helen Usansky, Sanjeeva Reddy, Louis J. Denis, Filip Janku. Phase 1 clinical safety and efficacy of ASN007, a novel oral ERK1/2 inhibitor, in patients with RAS, RAF or MEK mutant advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR09. doi:10.1158/1535-7163.TARG-19-PR09

Accumulative persistence of the genotoxic and mutagenic effects induced by low doses of TiO2 nanoparticles increases the incidence of hepatocellular carcinoma in mice

Nowadays, titanium dioxide nanoparticles (TiO2NPs) are widely used by many industries and are present in many products including toothpastes, sweets, and medications. Therefore, humans are exposed to considerable amounts of this material in their daily lives. However, limited studies are available regarding the relationship between the effects of TiO2NP-induced genotoxicity and the incidence of liver cancer. Therefore, this study was designed to investigate the impact of TiO2NPs-induced genotoxicity and mutagenicity in the livers of mice. Further, we assessed the development of hepatic cancer in these mice. Oral administration of TiO2NPs resulted in persistent single and double stranded DNA breaks which were indicated by the fragmented and smeared genomic DNA observed on an agarose gel. This damaged DNA resulted in apoptosis-induced liver damage. Progressive accumulation of TiO2NPs-induced DNA damage resulted in a high mutation incidence in both the p53 and H-ras genes. Moreover, histological examinations revealed necrosis throughout the liver during the experimental period. This damage could be attributed to the persistent and significant increase in malondialdehyde and reduction in glutathione levels. In conclusion, the accumulation of orally administered TiO2NPs resulted in persistent DNA damage, apoptosis, and oxidative stress. These events ultimately lead to necrosis in the entire liver, which increased the incidence of hepatocellular carcinoma.

Pharmacological biotargets and the molecular mechanisms of oxyresveratrol treating colorectal cancer: Network and experimental analyses.

This article was designed by using a network pharmacological approach to reveal the therapeutic targets and molecular mechanisms of oxyresveratrol (Oxyres) treating colorectal cancer (CRC). Furthermore, several bioinformatic findings would be validated. Pathogenetic targets of CRC and pharmacological targets of Oxyres were identified by web-available databases. All identifiable biotargets were collected for functional enrichment analyses to reveal the biological processes and signaling pathways of Oxyres treating CRC. In addition, human CRC, non-CRC samples, and cell line study were used to validate the predictive biotargets of Oxyres treating CRC. In network pharmacological analyses, top therapeutic targets of mitogen-activated protein kinase 1 (MAPK1), insulin growth factor 1 (IGF1), hematopoietic prostaglandin D synthase (HPGDS), GTPase HRas (HRAS), and cytochrome P450 2C9 (CYP2C9) in Oxyres treating CRC were identified, respectively. As shown in functional analysis, biological processes of Oxyres treating CRC were mainly involved in modulating cell communication, signal transduction, apoptosis, cell motility, cell proliferation, and lipid metabolism. Furthermore, top 10 signaling pathways of Oxyres treating CRC were identified, respectively. In human study, CRC samples resulted in increased neoplastic expressions of Ki-67, MAPK1, IGF1, characterized with clinical imaging inspection, pathological diagnosis, and altered blood lipids in these CRC cases. In cell culture study, Oxyres-dosed CRC cells exhibited reduced cell proliferation, promoted cellular apoptosis. Furthermore, significantly decreased proteins of intracellular Ki-67, MAPK1, and IGF1 were observed in Oxyres-dosed cells when compared to those in controls. Collectively, anti-CRC pharmacological activity of Oxyres may be mainly associated with induction of apoptosis and suppression of cell proliferation as revealed in bioinformatic findings. In addition, all core biotargets and molecular mechanisms of Oxyres treating CRC are unveiled respectively. Interestingly, the identifiable MAPK1, IGF1 biotargets may be potential molecules for treating and screening CRC.

A mutation analysis of the EGFR pathway genes, RAS, EGFR, PIK3CA, AKT1 and BRAF, and TP53 gene in thymic carcinoma and thymoma type A/B3.

Thymic carcinoma is rare and usually has a fatal outcome. Gene mutations in the epidermal growth factor receptor (EGFR) signalling pathway and TP53 have not been well analysed in thymic carcinoma. We examined a large cohort of thymic carcinoma and thymoma type A/B3 and looked for gene mutations in the RAS family, EGFR, PIK3CA, AKT1, BRAF and TP53. Among 54 thymic carcinoma cases, RAS family mutations were detected in 10 cases, EGFR in two, PIK3CA in one, AKT1 in one, BRAF in none and TP53 in five. Among 33 thymoma type A/B3 cases, HRAS gene mutation were found in one, PIK3CA in two and AKT1 in one. All these mutations were those of missense type activating mutations. RAS family mutations were significantly more frequent in thymic carcinoma than in thymoma type A/B3 (P=0.0461). A prognostic analysis focusing on thymic squamous cell carcinoma cases (n=44) showed that the overall survival was significantly shorter in patients with EGFR pathway mutations (n=9) than in those without in a univariate analysis (P=0.0173). Subsequently, EGFR pathway mutations were selected as an independent factor for a poor overall survival in a multivariate analysis (P=0.0389). Mutations in the EGFR pathway and TP53 in thymic carcinoma may be frequent, and the EGFR pathway mutations may be associated with a poor prognosis in thymic squamous cell carcinoma patients. The therapeutic significance of gene mutations in thymic carcinoma should be further clarified.

Short Call Abstracts

Short Call Abstracts

Dysregulated mitogen-activated protein kinase pathway mediated cell cycle disruption in sporadic parathyroid tumors.

The study was designed to evaluate expression profiling of mitogen-activated protein kinase (MAPK) signalling pathway genes in sporadic parathyroid adenoma. Expression of MAPK signalling pathway genes including activated transcription factors and cell cycle regulatory genes was analysed by real-time PCR- based array in parathyroid adenoma (N = 20) and normal parathyroid tissue (N = 4). MAPK signalling pathway as studied by PCR array revealed that a total of 22 genes were differentially expressed (≥ twofold change, p ≤ 0.05) in parathyroid adenoma. Up-regulated genes were ARAF, MAPK12, CREBBP, MYC, HSPB1, HRAS, CDK4, CCND1, and E2F1, and down-regulated genes were MAP4K1, DLK1, MAP3K4, MAPK10, MAPK8, ATF2, SMAD4, MEF2C, LAMTOR3, FOS, CDKN2A CDKN2B, and RB1. The present study revealed that ERK1/2 signalling pathway with up-regulation of HRAS, ARAF, and MEK1 genes and up-regulation of positive regulators of cell cycle (CCND1, CDK4, and E2F1) and down-regulation negative regulators of cell cycle (CDKN2A, CDKN2B, and RB1) made highly dysregulated MAPK signalling pathway in parathyroid adenoma. Expression of CDK4 was positively associated with plasma PTH level (r = 0.60, p = 0.04) and tumor weight (r = 0.80, p = 0.02) of the adenoma patients, respectively. Expression of CDKN2A was correlated negatively with PTH level (r = - 0.52, p = 0.04) of the adenoma patients. The current study revealed that ERK pathway and associated cell cycle regulator genes are dysregulated in sporadic parathyroid adenoma.

Sodium nitroprusside induces H-Ras depalmitoylation and alters the cellular response to hypoxia in differentiated and undifferentiated PC12 cells.

Ras-GTPases regulate many central signalling pathways in the cell. Hypoxia induces nitrosative/oxidative stress and dysregulates Ras-dependent downstream processes. H-Ras possesses two cysteine residues (C181 and C184) in the C-termini, which are palmitoylated once or twice. Palmitoylation is sufficient for promoting stable plasma membrane localization. We hypothesized that high concentrations of hypoxia-formed nitric oxide could induce terminal cysteine S-nitrosylation, followed by depalmitoylation and H-Ras mislocalization. We investigated the action of a 100-μM nitric oxide-donor (sodium nitroprusside [SNP]) and a 100-μM palmitoylation inhibitor (2-bromopalmitate) on the distribution of membrane-bound S-nitrosylated and palmitoylated H-Ras under hypoxic/normoxic conditions in undifferentiated/differentiated pheochromocytoma (PC12) cells. We found that under normoxic conditions, SNP increases membrane-bound H-Ras nitrosylation only in differentiated cells, whereas under hypoxic conditions, SNP stimulates H-Ras nitrosylation in both differentiated and undifferentiated cells. SNP greatly decreases the palmitoylation of H-Ras under hypoxic conditions in both undifferentiated and differentiated cells, while under normoxic conditions, the effect of SNP is more negligible. Furthermore, Western blot analyses have shown that SNP decreases ERK phosphorylation under hypoxic conditions, in parallel with an elevation in hypoxia-induced factor activity and intracellular succinate concentration. We propose that high concentrations of hypoxia-formed nitric oxide can nitrosylate H-Ras terminal cysteines, which induce H-Ras activity dysregulation and alter the cellular response to hypoxia. SIGNIFICANCE OF THE STUDY: To our knowledge, these observations may be important for cancer prevention and therapy because cancer is one of the most prevalent disorders caused by the misregulation of Ras activity by a redox agent. Oncogenic activation of the H-Ras gene has been found in a wide variety of neoplastic transformations, and thus, investigation of the redox regulation of H-Ras activity is significant for cancer research as well.

Computational approaches: discovery of GTPase HRas as prospective drug target for 1,3-diazine scaffolds

Heterocyclic 1,3-diazine nucleus is a valuable pharmacophore in the field of medicinal chemistry and exhibit a wide spectrum of biological activities. PharmMapper, a robust online tool used for establishing the target proteins based on reverse pharmacophore mapping. PharmMapper study is carried out to explore the pharmacological activity of 1,3-diazine derivatives using reverse docking program. PharmMapper, an open web server was used to recognize for all the feasible target proteins for the developed compounds through reverse pharmacophore mapping. The results were analyzed via molecular docking with maestro v11.5 (Schrodinger 2018-1) using GTPase HRas as possible target. The molecular docking studies displayed the binding behavior of 1,3-diazine within GTP binding pocket. From the docking study compounds s3 and s14 showed better docked score with anticancer potency against cancer cell line (HCT116). Hence, the GTPase HRas may be the possible target of 1,3-diazine derivatives for their anticancer activity where the retrieved information may be quite useful for developing rational drug designing. Furthermore the selected 1,3-diazine compounds were evaluated for their in vitro anticancer activity against murine macrophages cell line. 1,3-Diazine compounds exhibited good selectivity of the compounds towards the human colorectal carcinoma cell line instead of the murine macrophages. The toxicity study of the most active compounds was also performed on non cancerous HEK-293 cell line.

Distinct mutational landscape of inverted urothelial papilloma.

A recent study has identified gene mutations involving the MAPK/ERK pathway, particularly the HRAS gene, in all inverted urothelial papillomas (IUPs), in the absence of pathway mutations in TERT promoter, FGFR3, and TP53/RB1genes. Neither recurrence nor progression was observed in IUPs. These data support several longstanding hypotheses: (1) IUPs are benign and do not recur or progress; (2) they harbor mutations that are different from those of urothelial carcinoma; and (3) they arise through different molecular mechanisms than low- or high-grade urothelial carcinoma. As the most critical differential diagnosis in this context is inverted-type urothelial carcinoma, more comprehensive studies are needed to compare and contrast these entities. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 4455: Discovery of AMG 510, a first-in-human covalent inhibitor of KRASG12C for the treatment of solid tumors

Abstract The RAS gene family encodes the small GTPase proteins NRAS, HRAS, and KRAS, which play an essential role in cellular growth and proliferation. KRAS is one of the most frequently mutated oncogenes in human cancer, with KRAS p.G12D, p.G12V, and p.G12C constituting the major mutational subtypes across lung, colon, and pancreatic cancers. Despite more than three decades of research, indirect approaches targeting KRAS mutant cancers have largely failed to show clinical benefit, and direct approaches have been stymied by the apparently ‘undruggable’ nature of KRAS. Cysteine-12 of KRASG12C has recently emerged as a unique vulnerability in KRAS-mutant cancers, and a small number of cysteine-reactive inhibitory tool molecules have been disclosed. We here report independent efforts to identify cysteine-reactive molecules capable of selectively inhibiting KRASG12C. Through iterative screening and structural biology efforts, we identified a novel Cys12-reactive inhibitor scaffold that derived its potency from occupancy of a previously unknown cryptic pocket induced by side-chain motion of the His95 residue of KRAS. Employing a scaffold-hopping approach, we leveraged knowledge of this cryptic pocket to design a series of N-aryl quinazolin-2(1H)-one-based inhibitors that demonstrated significantly enhanced potency relative to prior tool compounds. Extensive optimization of these leads led to the identification of a highly potent, selective, and well-tolerated inhibitor of KRASG12C, which was nominated for clinical development as AMG 510. In preclinical tumor models, AMG 510 rapidly and irreversibly binds to KRASG12C, providing durable suppression of the mitogen-activated protein kinase (MAPK) signaling pathway. Dosed orally (once daily) as a single agent, AMG 510 is capable of inducing tumor regression in mouse models of KRASG12C cancer. AMG 510 is, to the best of our knowledge, the first direct KRASG12C therapeutic to reach human clinical testing and is currently in a Phase I clinical trial evaluating safety, tolerability, PK, and efficacy in subjects with solid tumors bearing the KRAS p.G12C mutation (NCT03600883). Citation Format: Brian A. Lanman, Jian Jeffrey Chen, Longbin Liu, Patricia Lopez, Alexander J. Pickrell, Anthony B. Reed, Hui-Ling Wang, Pragathi Achanta, Jude Canon, Daniel A. Erlanson, Raymond V. Fucini, Joon Won Jeong, Christopher Mohr, Anne Y. Saiki, Victor J. Cee, J. Russell Lipford, Karen Rex, Laurie P. Volak. Discovery of AMG 510, a first-in-human covalent inhibitor of KRASG12C for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4455.

Costello syndrome: Clinical phenotype, genotype, and management guidelines.

Costello syndrome (CS) is a RASopathy caused by activating germline mutations in HRAS. Due to ubiquitous HRAS gene expression, CS affects multiple organ systems and individuals are predisposed to cancer. Individuals with CS may have distinctive craniofacial features, cardiac anomalies, growth and developmental delays, as well as dermatological, orthopedic, ocular, and neurological issues; however, considerable overlap with other RASopathies exists. Medical evaluation requires an understanding of the multifaceted phenotype. Subspecialists may have limited experience in caring for these individuals because of the rarity of CS. Furthermore, the phenotypic presentation may vary with the underlying genotype. These guidelines were developed by an interdisciplinary team of experts in order to encourage timely health care practices and provide medical management guidelines for the primary and specialty care provider, as well as for the families and affected individuals across their lifespan. These guidelines are based on expert opinion and do not represent evidence-based guidelines due to the lack of data for this rare condition.

Network pharmacology-based identifcation of potential targets of the flower of Trollius chinensis Bunge acting on anti-inflammatory effectss

The flower of Trollius chinensis Bunge was widely used for the treatment of inflammation-related diseases in traditional Chinese medicine (TCM). In order to clarify the anti-inflammatory mechanism of this Chinese herbs, a comprehensive network pharmacology strategy that consists of three sequential modules (pharmacophore matching, enrichment analysis and molecular docking.) was carried out. As a result, Apoptosis signal-regulating kinase 1 (ASK1), Janus kinase 1 (JAK1), c-Jun N-terminal kinases (JNKs), transforming protein p21 (HRas) and mitogen-activated protein kinase 14 (p38α) that related to the anti-inflammatory effect were filtered out. In further molecular dynamics (MD) simulation, the conformation of CID21578038 and CID20055288 were found stable in the protein ASK1 and JNKs respectively. The current investigation revealed that two effective compounds in the flower of Trollius chinensis Bunge played a crucial role in the process of inflammation by targeting ASK1 and JNKs, the comprehensive strategy can serve as a universal method to guide in illuminating the mechanism of the prescription of traditional Chinese medicine by identifying the pathways or targets.

Mutation profiling of anaplastic ependymoma grade III by Ion Proton next generation DNA sequencing.

Background: Ependymomas are glial tumors derived from differentiated ependymal cells. In contrast to other types of brain tumors, histological grading is not a good prognostic marker for these tumors. In order to determine genomic changes in an anaplastic ependymoma, we analyzed its mutation patterns by next generation sequencing (NGS). Methods: Tumor DNA was sequenced using an Ion PI v3 chip on Ion Proton instrument and the data were analyzed by Ion Reporter 5.6. Results: NGS analysis identified 19 variants, of which four were previously reported missense variants; c.395G>A in IDH1, c.1173A>G in PIK3CA, c.1416A>T in KDR and c.215C>G in TP53. The frequencies of the three missense mutations ( PIK3CA c.1173A>G, KDR c.1416A>T, TP53, c.215C>G) were high, suggesting that these are germline variants, whereas the IDH1 variant frequency was low (4.81%). However, based on its FATHMM score of 0.94, only the IDH1 variant is pathogenic; other variants TP53, PIK3CA and KDR had FATHMM scores of 0.22, 0.56 and 0.07, respectively. Eight synonymous mutations were found in FGFR3, PDGFRA, EGFR, RET, HRAS, FLT3, APC and SMAD4 genes. The mutation in FLT3 p.(Val592Val) was the only novel variant found. Additionally, two known intronic variants in KDR were found and intronic variants were also found in ERBB4 and PIK3CA. A known splice site mutation at an acceptor site in FLT3, a 3'-UTR variant in the CSF1R gene and a 5'_UTR variant in the SMARCB1 gene were also identified. The p-values were below 0.00001 for all variants and the average coverage for all variants was around 2000x. Conclusions: In this grade III ependymoma, one novel synonymous mutation and one deleterious missense mutation is reported. Many of the variants reported here have not been detected in ependymal tumors by NGS analysis previously and we therefore report these variants in brain tissue for the first time.

Clinical impact of testing for mutations and microRNAs in thyroid nodules.

BackgroundWe report results of a multicenter clinical experience study examining the likelihood of patients with indeterminate thyroid nodules to undergo surgery or have malignant outcome based on multiplatform combination mutation and microRNA testing (MPT).MethodsMPT assessed mutations in BRAF, HRAS, KRAS, NRAS, and PIK3CA genes, PAX8/PPARγ, RET/PTC1, and RET/PTC3 gene rearrangements, and the expression of 10 microRNAs. Baseline clinical information at the time of MPT and clinical follow‐up records were reviewed for 337 patients, of which 80% had negative MPT results. Kaplan Meier analysis for cumulative probability of survival without having a surgical procedure or malignant diagnosis over the course of patient follow‐up was determined for MPT results of 180 patients, among which only 14% had malignancy.ResultsA negative MPT result in nodules with Bethesda III or IV cytology (2009) conferred a high probability of non‐surgical treatment, with only 11% expected to undergo surgery and a high probability of survival without malignancy (92%) for up to 2 years follow up. A positive MPT result conferred a 57% probability of malignancy and was an independent risk factor for undergoing surgical treatment (Hazard Ratio [HR] 9.2, 95% confidence intervals 5.4‐15.9, P < .0001) and for malignancy (HR 13.4, 95% confidence intervals 4.8‐37.2, P < .0001). For nodules with weak driver mutations, positive microRNA test results supported high risk of cancer while negative results downgraded cancer risk.ConclusionMPT results are predictive of real‐world decisions to surgically treat indeterminate thyroid nodules, with those decisions being appropriately aligned with a patient's risk of malignancy over time.

New insights into radioresistance in breast cancer identify a dual function of miR-122 as a tumor suppressor and oncomiR.

Radioresistance of tumor cells gives rise to local recurrence and disease progression in many patients. MicroRNAs (miRNAs) are master regulators of gene expression that control oncogenic pathways to modulate the radiotherapy response of cells. In the present study, differential expression profiling assays identified 16 deregulated miRNAs in acquired radioresistant breast cancer cells, of which miR‐122 was observed to be up‐regulated. Functional analysis revealed that miR‐122 has a role as a tumor suppressor in parental cells by decreasing survival and promoting radiosensitivity. However, in radioresistant cells, miR‐122 functions as an oncomiR by promoting survival. The transcriptomic landscape resulting from knockdown of miR‐122 in radioresistant cells showed modulation of the ZNF611, ZNF304, RIPK1, HRAS, DUSP8 and TNFRSF21 genes. Moreover, miR‐122 and the set of affected genes were prognostic factors in breast cancer patients treated with radiotherapy. Our data indicate that up‐regulation of miR‐122 promotes cell survival in acquired radioresistant breast cancer and also suggest that miR‐122 differentially controls the response to radiotherapy by a dual function as a tumor suppressor an and oncomiR dependent on cell phenotype.

Expansion of the phenotypic spectrum and description of molecular findings in a cohort of patients with oculocutaneous mosaic RASopathies.

BackgroundPostzygotic KRAS, HRAS, NRAS, and FGFR1 mutations result in a group of mosaic RASopathies characterized by related developmental anomalies in eye, skin, heart, and brain. These oculocutaneous disorders include oculoectodermal syndrome (OES) encephalo‐cranio‐cutaneous lipomatosis (ECCL), and Schimmelpenning‐Feuerstein‐Mims syndrome (SFMS). Here, we report the results of the clinical and molecular characterization of a novel cohort of patients with oculocutaneous mosaic RASopathies.MethodsTwo OES, two ECCL, and two SFMS patients were ascertained in the study. In addition, two subjects with unilateral isolated epibulbar dermoids were also enrolled. Molecular analysis included PCR amplification and Sanger sequencing of KRAS, HRAS, NRAS, and FGFR1 genes in DNA obtained from biopsies (skin/epibulbar dermoids), buccal mucosa, and blood leukocytes. Massive parallel sequencing was employed in two cases with low‐level mosaicism.ResultsIn DNA from biopsies, mosaicism for pathogenic variants, including KRAS p.Ala146Thr in two OES subjects, FGFR1 p.Asn546Lys and KRAS p.Ala146Val in ECCL patients, and KRAS p.Gly12Asp in both SFMS patients, was demonstrated. No mutations were shown in DNA from conjunctival lesions in two subjects with isolated epibubar dermoids.ConclusionOur study allowed the expansion of the clinical spectrum of mosaic RASopathies and supports that mosaicism for recurrent mutations in KRAS and FGFR1 is a commonly involved mechanism in these rare oculocutaneous anomalies.

Assessment of HMGA2 and PLAG1 rearrangements in breast adenomyoepitheliomas

Breast adenomyoepitheliomas (AMEs) are rare epithelial-myoepithelial neoplasms that may occasionally produce myxochondroid matrix, akin to pleomorphic adenomas (PAs). Regardless of their anatomic location, PAs often harbor rearrangements involving HMGA2 or PLAG1. We have recently shown that the repertoire of somatic genetic alterations of AMEs varies according to their estrogen receptor (ER) status; whilst the majority of ER-positive AMEs display mutually exclusive PIK3CA or AKT1 hotspot mutations, up to 60% of ER-negative AMEs harbor concurrent HRAS Q61 hotspot mutations and mutations affecting either PIK3CA or PIK3R1. Here, we hypothesized that a subset of AMEs lacking these somatic genetic alterations could be underpinned by oncogenic fusion genes, in particular those involving HMGA2 or PLAG1. Therefore, we subjected 13 AMEs to RNA-sequencing for fusion discovery (n = 5) and/or fluorescence in situ hybridization (FISH) analysis for HMGA2 and PLAG1 rearrangements (n = 13). RNA-sequencing revealed an HMGA2-WIF1 fusion gene in an ER-positive AME lacking HRAS, PIK3CA and AKT1 somatic mutations. This fusion gene, which has been previously described in salivary gland PAs, results in a chimeric transcript composed of exons 1–5 of HMGA2 and exons 3–10 of WIF1. No additional in-frame fusion genes or HMGA2 or PLAG1 rearrangements were identified in the remaining AMEs analyzed. Our results demonstrate that a subset of AMEs lacking mutations affecting HRAS and PI3K pathway-related genes may harbor HMGA2-WIF1 fusion genes, suggesting that a subset of breast AMEs may be genetically related to PAs or that a subset of AMEs may originate in the context of a PA.

Inverse Molecular Docking as a Novel Approach to Study Anticarcinogenic and Anti-Neuroinflammatory Effects of Curcumin.

Research efforts are placing an ever increasing emphasis on identifying signal transduction pathways related to the chemopreventive activity of curcumin. Its anticarcinogenic effects are presumably mediated by the regulation of signaling cascades, including nuclear factor κB (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinases (MAPK). By modulating signal transduction pathways, curcumin induces apoptosis in malignant cells, thus inhibiting cancer development and progression. Due to the lack of mechanistic insight in the scientific literature, we developed a novel inverse molecular docking protocol based on the CANDOCK algorithm. For the first time, we performed inverse molecular docking of curcumin into a collection of 13,553 available human protein structures from the Protein Data Bank resulting in prioritized target proteins of curcumin. Our predictions were in agreement with the scientific literature and confirmed that curcumin binds to folate receptor β, DNA (cytosine-5)-methyltransferase 3A, metalloproteinase-2, mitogen-activated protein kinase 9, epidermal growth factor receptor and apoptosis-inducing factor 1. We also identified new potential protein targets of curcumin, namely deoxycytidine kinase, NAD-dependent protein deacetylase sirtuin-1 and -2, ecto-5′-nucleotidase, core histone macro-H2A.1, tyrosine-protein phosphatase non-receptor type 11, macrophage colony-stimulating factor 1 receptor, GTPase HRas, aflatoxin B1 aldehyde reductase member 3, aldo-keto reductase family 1 member C3, amiloride-sensitive amine oxidase, death-associated protein kinase 2 and tryptophan-tRNA ligase, that may all play a crucial role in its observed anticancer effects. Moreover, our inverse docking results showed that curcumin potentially binds also to the proteins cAMP-specific 3′,5′-cyclic phosphodiesterase 4D and 17-β-hydroxysteroid dehydrogenase type 10, which provides a new explanation for its efficiency in the treatment of Alzheimer’s disease. We firmly believe that our computational results will complement and direct future experimental studies on curcumin’s anticancer activity as well as on its therapeutic effects against Alzheimer’s disease.